1. Field of the Invention
This invention relates to a carrier adapted for fixation of a physiologically active substance, and a process for producing the carrier. This invention also relates to a bioreactor comprising the carrier. This invention further relates to a chip for surface plasmon resonance analysis.
2. Description of the Related Art
Various analyses utilizing intermolecular interactions, such as immune reactions, have heretofore been performed in the fields of clinical examinations, and the like. Among others, several kinds of techniques, which do not require complicated operations and labeling substances and which are capable of detecting alterations in binding quantities of analyzed substances with a high sensitivity, have heretofore been used. Examples of the techniques described above include surface plasmon resonance (SPR) analysis techniques, quartz crystal oscillator microbalance (QCM) analysis techniques, and analysis techniques utilizing functional surfaces, such as surfaces of gold colloidal particles and ultrafine particles. In each of the techniques described above, a surface for fixation of a physiologically active substance is important. By way of example, the surface plasmon resonance (SPR) analysis techniques will be described hereinbelow.
Ordinarily, an analysis chip for use in analysis of a physiologically active substance is provided with a transparent base plate (e.g., a glass plate), a metal film, which has been formed on the transparent base plate by use of a vacuum evaporation processing, and a thin film, which has been formed on the metal film and which has a functional group capable of fixing a physiologically active substance, such as a protein. The physiologically active substance is fixed to the metal surface via the functional group. A specific binding reaction between the physiologically active substance and a sample substance is analyzed, and an interaction between biomolecules is thereby analyzed.
There have been known several techniques for fixation of a physiologically active substance to an analysis chip. For example, in cases where the physiologically active substance is a protein, as a technique for fixing the analysis chip and the protein with each other through covalent bonding, there has been known a technique (i.e., an amine coupling technique), wherein an amino group of the protein and a carboxyl group on the analysis chip are bound with each other. However, with the amine coupling technique, since an arbitrary amino group on the surface of the protein is modified due to the fixation, it often occurs that the orientation of fixed protein is not capable of coinciding with a predetermined orientation, or it often occurs that the binding of the protein and the substrate with each other is obstructed by the position of the modified amino group, and that the activity of the protein becomes low. Also, with the amine coupling technique, it is necessary for the protein to be concentrated on the analysis chip, and it is necessary that, at the time of the fixation, the protein is dissolved in a buffer solution, which has a pH value lower than pI of the protein to be fixed and which has a low ionic strength. Therefore, in the cases of a protein which undergoes denaturation under the conditions described above, the problems occur in that the fixation of the protein is not capable of being performed with the activity of the protein being kept.
Also, there have been developed techniques, wherein a protein is fixed onto an analysis chip under neutral conditions by use of a part referred to as a tag, which has been introduced to an N terminal or a C terminal of a protein having been synthesized artificially through generic alteration. A typical example of the technique described above is a fixation technique utilizing His-tag. The fixation technique utilizing the His-tag has been developed for an affinity column for purification of a His-tag protein having been expressed through genetic recombination. The fixation technique utilizing the His-tag has also been used for fixing a protein onto a solid surface such that the protein may have predetermined orientational characteristics.
Particularly, with a technique for fixation of the His-tag protein, wherein an NTA-Ni(II) complex having been formed from nitrilotriacetic acid (NTA) and an Ni(II) ion is utilized, water molecules having coordinated with two coordinating dentations in the complex are substituted by nitrogen atoms of two imidazole groups of an oligohistidine residue of the His-tag protein, and the His-tag protein is thereby bound with the solid surface specifically and in a predetermined direction. With the technique for fixation of the His-tag protein, wherein the NTA-Ni(II) complex is utilized, since pre-concentration under acidic conditions need not be performed, the fixation of the His-tag protein by use of a buffer solution (such as PBS) under physiological conditions is capable of being performed, and the problems encountered with the amine coupling technique are capable of being eliminated.
However, since the combination of the His-tag protein and the NTA-Ni(II) complex with each other has been developed for the purposes of the purification with the affinity column, the binding between the His-tag protein and the NTA-Ni(II) complex is not sufficiently strong, and the problems with regard to dissociation equilibrium are encountered. Therefore, the problems occur in that the His-tag protein having been fixed via the NTA-Ni(II) complex onto the analysis chip undergoes dissociation little by little from the analysis chip. Accordingly, the combination of the His-tag protein and the NTA-Ni(II) complex with each other is not capable of being applied directly to the use applications for biosensors, and the like.
Several studies have been made for solving the problems with regard to the dissociation described above. For example, fixation techniques, wherein substitution inactivation of a metal ion coordinating with the His-tag protein is effected through oxidation with an oxidizing agent, or the like, are disclosed in Japanese Unexamined Patent Publication No. 2006-266831 and U.S. Pat. No. 5,439,829 corresponding to Japanese Unexamined Patent Publication No. 6(1994)-157600. However, with the disclosed fixation techniques, the problems often occur, depending upon the oxidation rate and the kind of the oxidizing agent, in that deactivation of the protein arises. Also, an attempt for improving the binding by the utilization of triNTA, in lieu of NTA described above, as a ligand is described in, for example, International Patent Publication No. WO00/047548. However, with the attempt for improving the binding by the utilization of triNTA, it is not always possible to obtain practically sufficient fixation.
A technique for fixing NTA to a polysaccharide is disclosed in, for example, Farid Khan et al., “Double-Hexahistidine Tag with High-Affinity Binding for Protein Immobilization, Purification, and Detection on Ni-Nitrilotriacetic Acid Surfaces”, Analytical Chemistry, Vol. 78, No. 9, pp. 3072-3079, 2006. Also, a technique for fixation of the His-tag protein utilizing the NTA-Ni(II) complex, wherein the imidazole groups of the His-tag protein and NI(II) are bound at multiple points with the NTA ligand, is disclosed in, for example, Suman Late and Jacob Piehler, “Stable and Functional Immobilization of Histidine-Tagged Proteins via Multivalent Chelator Headgroups on a Molecular Poly(ethylene glycol) Brush”, Analytical Chemistry, Vol. 77, No. 4, pp. 1096-1105, 2005.
It may be presumed that, in cases where the physiologically active substance is capable of being supported at multiple points, the binding force will be capable of being enhanced, and the aforesaid problems with regard to the dissociation will be capable of being solved. However, in Farid Khan et al., “Double-Hexahistidine Tag with High-Affinity Binding for Protein Immobilization, Purification, and Detection on Ni-Nitrilotriacetic Acid Surfaces”, Analytical Chemistry, Vol. 78, No. 9, pp. 3072-3079, 2006, and Suman Lata and Jacob Piehler, “Stable and Functional Immobilization of Histidine-Tagged Proteins via Multivalent Chelator Headgroups on a Molecular Poly(ethylene glycol) Brush”, Analytical Chemistry, Vol. 77, No. 4, pp. 1096-1105, 2005, nothing is studied with respect to a level of an NTA density, with which the problems with regard to the dissociation will be capable of being solved. Also, with the fixation technique described in Suman Late and Jacob Piehler, “Stable and Functional Immobilization of Histidine-Tagged Proteins via Multivalent Chelator Headgroups on a Molecular Poly(ethylene glycol) Brush”, Analytical Chemistry, Vol. 77, No. 4, pp. 1096-1105, 2005, since the ligands close to each other are rigid and are not capable of moving flexibly, the problems are encountered in that the metal is not always capable of coordinating at multiple points with the protein, and in that actually it is not always possible to fix the protein reliably at multiple points.